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• W1965458421 abstract "The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs. The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs. The fibroblast growth factors (FGFs) 5The abbreviations used are: FGF, fibroblast growth factor; FGFR1, FGF receptor-1; FGFR1-ST, extracellular domain of FGFR1-IIIc with a C-terminal Strep-Tag II; GPI, glycosylphosphatidylinositol; FGFR1-GPI, extracellular domain of FGFR1-IIIc with a C-terminal GPI anchor; MALDI-TOF, matrix-assisted laser desorption /ionization-time of flight; PBS, phosphate-buffered saline; QCM-D, quartz crystal microbalance-dissipation; PDB, Protein Data Bank; FACS, fluorescent-activated cell sorting; CHO, Chinese hamster ovary; HS, heparan sulfate. and their receptors co-evolved with metazoans (1Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 1-12Crossref Google Scholar, 2Itoh N. Ornitz D.M. Trends Genet. 2004; 20: 563-569Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar), a reflection of their central role in mediating cell-cell communication. FGFs regulate the specification of stem cell fate, organogenesis, skeletal growth (3Ornitz D.M. Cytokine Growth Factor Rev. 2005; 16: 205-213Crossref PubMed Scopus (296) Google Scholar), tissue repair (4Detillieux K.A. Sheikh F. Kardami E. Cattini P.A. Cardiovasc. Res. 2003; 57: 8-19Crossref PubMed Scopus (189) Google Scholar) and phosphate metabolism. FGFs are involved in numerous pathologies such as genetic skeletal defects in children (5Ornitz D.M. Marie P.J. Genes Dev. 2002; 16: 1446-1465Crossref PubMed Scopus (726) Google Scholar) and carcinomas in adults (6Freeman K.W. Gangula R.D. Welm B.E. Ozen M. Foster B.A. Rosen J.M. Ittmann M. Greenberg N.M. Spencer D.M. Cancer Res. 2003; 63: 6237-6243PubMed Google Scholar, 7van Rhijn B.W. Lurkin I. Radvanyi F. Kirkels W.J. van der Kwast T.H. Zwarthoff E.C. Cancer Res. 2001; 61: 1265-1268PubMed Google Scholar). In humans and rodents there are 22 genes encoding more than 30 different FGF proteins and the fgfr 1-4 genes encode over 48 major isoforms of the cognate receptor tyrosine kinase (FGFR) (1Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 1-12Crossref Google Scholar, 2Itoh N. Ornitz D.M. Trends Genet. 2004; 20: 563-569Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar, 8Groth C. Lardelli M. Int. J. Dev. Biol. 2002; 46: 393-400PubMed Google Scholar). FGFs possess a glycosaminoglycan co-receptor, usually heparan sulfate (HS). The interaction of FGFs with HS is required for the stimulation of cell proliferation, which is initiated by the formation of a complex of FGF ligand, HS co-receptor and FGFR (9Delehedde M. Seve M. Sergeant N. Wartelle I. Lyon M. Rudland P.S. Fernig D.G. J. Biol. Chem. 2000; 275: 33905-33910Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Delehedde M. Lyon M. Sergeant N. Rahmoune H. Fernig D.G. J. Mammary Gland Biol. Neoplasia. 2001; 6: 253-273Crossref PubMed Scopus (62) Google Scholar, 11Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1503) Google Scholar). Not surprisingly for a family of key regulatory ligands, the activation of FGF signaling is subjected to multiple regulatory inputs, some of which operate at the level of the assembly of the receptor-ligand complex, whereas others operate inside the cell, on the active receptor complex. Regulatory inputs that operate extracellularly include partial selectivity in the choice of ligand by certain receptor isoforms. FGF-7 is the most specific FGF ligand, because it only interacts with the IIIb isoform of FGFR2; other FGFs show varying degrees of promiscuity in terms of their FGFR preference (12Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1423) Google Scholar). The HS chain mediates a second extracellular regulatory input. Specific sequences in HS can allow only a restricted subset of FGF-FGFR interactions to lead to cell proliferation (13Ostrovsky O. Berman B. Gallagher J. Mulloy B. Fernig D.G. Delehedde M. Ron D. J. Biol. Chem. 2002; 277: 2444-2453Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 14Rahmoune H. Gallagher J.T. Rudland P.S. Fernig D.G. J. Biol. Chem. 1998; 273: 7303-7310Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Merry C.L. Bullock S.L. Swan D.C. Backen A.C. Lyon M. Beddington R.S. Wilson V.A. Gallagher J.T. J. Biol. Chem. 2001; 276: 35429-35434Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 16Guimond S.E. Turnbull J.E. Curr. Biol. 1999; 9: 1343-1346Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) and this is likely to occur in vivo (17Kato M. Wang H. Kainulainen V. Fitzgerald M.L. Ledbetter S. Ornitz D.M. Bernfield M. Nat. Med. 1998; 4: 691-697Crossref PubMed Scopus (287) Google Scholar, 18Chang Z. Meyer K. Rapraeger A.C. Friedl A. Faseb J. 2000; 14: 137-144Crossref PubMed Scopus (94) Google Scholar). A third extracellular regulatory input is likely to be the assembly of the FGF receptor ligand system into different complexes that have different signaling potentials. This is suggested by the identification of two different crystal structures of complexes of FGFR·FGF·heparin oligosaccharide (19Pellegrini L. Burke D.F. von Delft F. Mulloy B. Blundell T.L. Nature. 2000; 407: 1029-1034Crossref PubMed Scopus (628) Google Scholar, 20Schlessinger J. Plotnikov A.N. Ibrahimi O.A. Eliseenkova A.V. Yeh B.K. Yayon A. Linhardt R.J. Mohammadi M. Mol. Cell. 2000; 6: 743-750Abstract Full Text Full Text PDF PubMed Scopus (964) Google Scholar) and by biophysical (21Harmer N.J. Ilag L.L. Mulloy B. Pellegrini L. Robinson C.V. Blundell T.L. J. Mol. Biol. 2004; 339: 821-834Crossref PubMed Scopus (95) Google Scholar) and biochemical evidence (9Delehedde M. Seve M. Sergeant N. Wartelle I. Lyon M. Rudland P.S. Fernig D.G. J. Biol. Chem. 2000; 275: 33905-33910Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 22Delehedde M. Lyon M. Gallagher J.T. Rudland P.S. Fernig D.G. Biochem. J. 2002; 366: 235-244Crossref PubMed Google Scholar). Finally, genetic evidence indicates that mutation of an asparagine residue in the extracellular domain of FGFR2 and FGFR3 increases its activity, to result in skeletal growth defects, which has been linked to the disruption of N-glycosylation (23Winterpacht A. Hilbert K. Stelzer C. Schweikardt T. Decker H. Segerer H. Spranger J. Zabel B. Physiol. Genomics. 2000; 2: 9-12Crossref PubMed Scopus (56) Google Scholar, 24Steinberger D. Mulliken J.B. Muller U. Hum. Mutat. 1996; 8: 386-390Crossref PubMed Scopus (25) Google Scholar). The latter observations suggest that N-glycosylation of FGFR may constitute a fourth regulatory input. To ascertain whether N-glycans on FGFR may regulate aspects of the assembly of the FGF ligand-receptor complex, the extracellular ligand binding domain of FGFR1-IIIc was expressed in Chinese Hamster Ovary (CHO) cells. We observed that it was heavily N-glycosylated and that multiple glycoforms of the receptor protein existed. The N-glycans were found to inhibit the interaction between FGFR1 and its FGF-2 ligand and HS co-receptor. The results suggest that N-glycosylation of FGFR1 represents an important additional pathway for the regulation of activity of the FGF receptor. Materials—Restriction enzymes and Vent polymerase were from New England Biolabs (Wilbury Way Hitchin, Herts, UK). Lipofectamine and Ni-NTA-agarose resin were from Invitrogen. CHO cells were the generous gift of Dr. D. Moss, University of Liverpool and were cultured in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 Ham (DMEM/F-12 Ham) supplemented with 5% (v/v) fetal calf serum (Invitrogen). Recombinant human FGF-2 was produced as described (25Ke Y. Wilkinson M.C. Fernig D.G. Smith J.A. Rudland P.S. Barraclough R. Biochim. Biophys. Acta. 1992; 1131: 307-310Crossref PubMed Scopus (32) Google Scholar). Heparin-derived dodecasaccharides were prepared from partial heparinase I digests of pig mucosal heparin and were obtained from Iduron (Manchester, UK). Bis[sulfosuccinimidyl] suberate, BS3, was from Perbio (Chester, UK). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, EDC, was from Fluka (Buchs, Switzerland). Thrombin, streptavidin, 11-mercaptoundecanoic acid, ethanolamine, hydrazine monohydrate and antibody to His6 were from Sigma. Strep-Tactinhorseradish peroxidase and Strep-Tactin-Sepharose resin were from IBA (Goettingen, Germany). Factor Xa was purchased from Roche Diagnostics (Lewes, East Sussex, UK) and N-glycanase (peptide N-glycosidase F) from Prozyme (Ely, UK). Plasmid Constructions—Two recombinant forms of the extracellular ligand binding domain of rat FGFR1-IIIc (26Yan G.C. Wang F. Fukabori Y. Sussman D. Hou J.Z. Mckeehan W.L. Biochem. Biophys. Res. Commun. 1992; 183: 423-430Crossref PubMed Scopus (41) Google Scholar) (amino acids 120-368 comprising the acid box and immunoglobulin loops D2 and D3) were produced. A soluble form of the receptor possessed a poly-histidine tail (His6) sequence followed by a thrombin cleavage site at the N terminus and a Factor Xa cleavage site followed by a Strep-Tag II sequence (27Schmidt T.G. Koepke J. Frank R. Skerra A. J. Mol. Biol. 1996; 255: 753-766Crossref PubMed Scopus (271) Google Scholar) at the C terminus and is termed FGFR1-ST (Fig. 1A). A membrane-bound form of the receptor possessed the same N-terminal His6 sequence and thrombin cleavage site, but after the Factor Xa cleavage site a GPI anchor attachment sequence replaced the Strep-Tag II sequence. This form of the receptor is termed FGFR1-GPI (Fig. 1A). Constructs encoding these two proteins were obtained using as a template pcDNA-h-fgfr1-gpi (gift from Dr. M. Seve, University of Liverpool) which corresponds to the rat fgfr1-IIIc sequence (amino acids 120-368) flanked by secretion signal and polyhistidine (His6) sequences in 5′ and a sequence encoding a GPI attachment sequence in 3′ (28Lodge A.P. Howard M.R. McNamee C.J. Moss D.J. Brain. Res. Mol. Brain. Res. 2000; 82: 84-94Crossref PubMed Scopus (45) Google Scholar). By performing two-step polymerase chain reactions (PCR) (29Horton R.M. Pease L.R. McPherson M.J. Directed Mutagenesis, A Practical Approach. IRL Press, Oxford1991Google Scholar) using sets of appropriate primers, sequences encoding cleavage sites for thrombin and for Factor Xa, as well as Strep-Tag II were introduced (Fig. 1B). Expression and Purification of FGFR1-ST and FGFR1-GPI Proteins—pcDNA-ht-fgfr1-xgpi and pcDNA-ht-fgfr1-xst constructs were linearized by digestion with BglII and transfected into CHO cells using Lipofectamine. Transfectants were selected by resistance to G418. For the soluble FGFR1-ST protein, clones expressing around 1 μg of receptor protein/ml culture medium were identified after plating into 96-well plates and Western blotting of culture medium with anti-His6. For the membrane-bound FGFR1-GPI, FACS using a Coulter Epics Altra flow cytometer (Beckman Coulter, Fullerton CA) was used to isolate a population of cells expressing the highest levels of receptor protein by labeling the cells with anti-His6 antibody and a second FITC-conjugated antibody. The population of cells identified and sorted by FACS were then plated into 96-well plates and clones expressing the highest levels of FGFR1-GPI were identified by Western blotting of cell extracts. The highest levels of expression of receptor protein for all clones was obtained by growing the cells in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 Ham supplemented with 0.5% (v/v) fetal calf serum for 72 h. In some experiments cells were cultured in the presence of 3 μg/ml tunicamycin for 24-48 h and recombinant proteins analyzed by SDS-PAGE followed by Western blot. For FGFR1-ST protein expression, cells were cultured in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 Ham supplemented with 0.5% (v/v) fetal calf serum in a rotor cell 12 prototype (Powell Brothers Ltd., Ormskirk, Lancs, UK). Medium was collected every 5 days. After flow dialysis of the culture medium, FGFR1-ST was purified by nickel chelation and Strep-Tactin-Sepharose chromatography. Vivaspin filtration unit (cut-off 30 kDa, Vivascience) was then used to concentrate and exchange the purified FGFR1-ST into PBS (phosphate-buffered saline, 140 mm NaCl, 10 mm Na2HPO4/NaH2PO4, pH 7.2). Concentration of receptor protein was determined by measurement of the absorbance at 280 nm. Removal of N-glycans of purified protein was performed by N-glycanase digestion in PBS buffer following the manufacturer's recommendations. To ensure equal amounts of protein were used in all experiments, a single aliquot of receptor protein was divided into two, one was treated with N-glycanase and the other mock-treated. Optical Biosensor Binding Assays—FGF-2 was immobilized on aminosilane surfaces using bissulfosuccinimidyl suberate (BS3 as the cross linker following the manufacturer's recommendations (NeoSensors, Sedgefield, UK). No more than 300 arc s FGF-2 was immobilized on the surface (1 arc s = 1/3600°, 600 arc s = 1 ng protein/mm2). Heparin-derived dodecasaccharides were biotinylated at their reducing ends and immobilized on streptavidin surfaces, as described (22Delehedde M. Lyon M. Gallagher J.T. Rudland P.S. Fernig D.G. Biochem. J. 2002; 366: 235-244Crossref PubMed Google Scholar, 30Delehedde M. Lyon M. Vidyasagar R. McDonnell T.J. Fernig D.G. J. Biol. Chem. 2002; 277: 12456-12462Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Binding assays were carried out in PBS supplemented with 0.02% (v/v) Tween 20 (PBST) at 20 °C following previously described methods (13Ostrovsky O. Berman B. Gallagher J. Mulloy B. Fernig D.G. Delehedde M. Ron D. J. Biol. Chem. 2002; 277: 2444-2453Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 14Rahmoune H. Gallagher J.T. Rudland P.S. Fernig D.G. J. Biol. Chem. 1998; 273: 7303-7310Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 22Delehedde M. Lyon M. Gallagher J.T. Rudland P.S. Fernig D.G. Biochem. J. 2002; 366: 235-244Crossref PubMed Google Scholar, 30Delehedde M. Lyon M. Vidyasagar R. McDonnell T.J. Fernig D.G. J. Biol. Chem. 2002; 277: 12456-12462Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31Powell A.K. Fernig D.G. Turnbull J.E. J. Biol. Chem. 2002; 277: 28554-28563Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 32West D.C. Rees C.G. Duchesne L. Patey S.J. Terry C.J. Turnbull J.E. Delehedde M. Heegaard C.W. Allain F. Vanpouille C. Ron D. Fernig D.G. J. Biol. Chem. 2005; 280: 13457-13464Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 33Fernig D.G. Iozzo R.V. Proteoglycan Protocols. Humana Press, Totowa, NJ2001Google Scholar) with minor modifications. A single binding assay consisted of adding the soluble binding partner or ligate in 1-3 μl PBST to a cuvette containing 22-24 μl or 37-39 μl PBST. The association reaction was followed until binding was at least 90% of the calculated equilibrium value, usually between 150 s and 230 s. The cuvette was then washed three times with 50 μl of PBST to initiate the dissociation of bound FGFR1-ST. The FGF-2 and oligosaccharide-derivatized surfaces were regenerated by washing twice with 50 μl of 2 m NaCl in 10 mm sodium phosphate buffer (10 mm Na2HPO4/NaH2P04, pH 7.2), PBST and 20 mm HCl, which removed 98-100% of bound FGFR1-ST. Binding parameters were calculated using the non-linear curve fitting program FASTFit (NeoSensors). Each binding assay yielded four binding parameters, which are the slope of initial rate of association, the on-rate constant (kon) and the extent of binding, all calculated from the association phase, and the off-rate constant (koff, equivalent to the dissociation rate constant, kd), calculated from the dissociation phase (13Ostrovsky O. Berman B. Gallagher J. Mulloy B. Fernig D.G. Delehedde M. Ron D. J. Biol. Chem. 2002; 277: 2444-2453Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 14Rahmoune H. Gallagher J.T. Rudland P.S. Fernig D.G. J. Biol. Chem. 1998; 273: 7303-7310Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 22Delehedde M. Lyon M. Gallagher J.T. Rudland P.S. Fernig D.G. Biochem. J. 2002; 366: 235-244Crossref PubMed Google Scholar, 30Delehedde M. Lyon M. Vidyasagar R. McDonnell T.J. Fernig D.G. J. Biol. Chem. 2002; 277: 12456-12462Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31Powell A.K. Fernig D.G. Turnbull J.E. J. Biol. Chem. 2002; 277: 28554-28563Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 32West D.C. Rees C.G. Duchesne L. Patey S.J. Terry C.J. Turnbull J.E. Delehedde M. Heegaard C.W. Allain F. Vanpouille C. Ron D. Fernig D.G. J. Biol. Chem. 2005; 280: 13457-13464Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 33Fernig D.G. Iozzo R.V. Proteoglycan Protocols. Humana Press, Totowa, NJ2001Google Scholar). All biosensor experiments were carried out at least two times on at least two different surfaces. Data Analysis—The determination of binding kinetics in optical biosensors can be prone to artifactual second phase binding sites, due either to rates of diffusion of soluble ligate approaching or exceeding the rate of association, or to steric hindrance at the binding surface (33Fernig D.G. Iozzo R.V. Proteoglycan Protocols. Humana Press, Totowa, NJ2001Google Scholar). Binding assays were designed to avoid such artifacts (13Ostrovsky O. Berman B. Gallagher J. Mulloy B. Fernig D.G. Delehedde M. Ron D. J. Biol. Chem. 2002; 277: 2444-2453Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 14Rahmoune H. Gallagher J.T. Rudland P.S. Fernig D.G. J. Biol. Chem. 1998; 273: 7303-7310Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 22Delehedde M. Lyon M. Gallagher J.T. Rudland P.S. Fernig D.G. Biochem. J. 2002; 366: 235-244Crossref PubMed Google Scholar, 30Delehedde M. Lyon M. Vidyasagar R. McDonnell T.J. Fernig D.G. J. Biol. Chem. 2002; 277: 12456-12462Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31Powell A.K. Fernig D.G. Turnbull J.E. J. Biol. Chem. 2002; 277: 28554-28563Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 32West D.C. Rees C.G. Duchesne L. Patey S.J. Terry C.J. Turnbull J.E. Delehedde M. Heegaard C.W. Allain F. Vanpouille C. Ron D. Fernig D.G. J. Biol. Chem. 2005; 280: 13457-13464Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 33Fernig D.G. Iozzo R.V. Proteoglycan Protocols. Humana Press, Totowa, NJ2001Google Scholar). Thus, limiting amounts of ligand were immobilized on the sensor surface, whereas the slope of initial rate, kon and the extent of binding were only determined at low concentrations of ligate, and koff was measured at higher concentrations of ligate, to avoid steric hindrance and rebinding artifacts. A single site model fitted the data at least as well as more complex models, was covered by at least 90% of the data, and, therefore, was used to calculate all binding parameters. The equilibrium dissociation constant (Kd) was calculated both from the ratio of the kd and ka and from the extent of binding, to provide an estimate of the self-consistency of the results. Quartz Crystal Microbalance-Dissipation (QCM-D)—A self-assembled monolayer was formed on a QCM-D (Q-Sense AB, Västra Frölunda, Sweden) gold crystal by overnight incubation in 11-mercaptoundecanoic acid (0.1 m in ethanol). The surface was then washed with ethanol, water (MilliQ) and then finally with PBS. All subsequent reactions were carried out in PBS. A hydrazide surface was formed by activating the terminal carboxylic acids with a wash of 300 μl of 0.25 m EDC followed by incubation in 300 μl of 0.2 m hydrazine monohydrate and 0.25 m EDC for 1 h. The surface was then washed vigorously with PBS. To immobilize a heparin-derived tetradecasaccharide, the hydrazide surface was incubated overnight in 300 μlof5 μg/ml heparin-derived tetradecasaccharide, a kind gift of Dr A. K. Powell, University of Liverpool. FGF-2 was immobilized on the hydrazide surface by cross-linking with BS3: 3 injections of 300 μl, 0.1 m BS3 over 10 min, followed by two injections of 300 μl, 37 μg/ml FGF-2. After the latter injection, the surface was incubated for 50 min before any unreacted BS3 was blocked with ethanolamine, 0.1 m, 300 μl, for 10 min. In a binding experiment, 300 μl of the deglycosylated or glycosylated FGFR1-ST (48.5 nm) was introduced into the cell and left to incubate for 10 min at 20 °C. The surface was then washed with PBS. The amount of FGFR1-ST bound was determined by comparing the frequency response before and after the addition of the receptor. This frequency change was converted into a mass change by using the Sauerbrey equation (34Sauerbrey G. Zeitschrift Fur Physik. 1959; 155: 206-222Crossref Scopus (9197) Google Scholar), with a conversion co-efficient of 17.7 ng cm-2 Hz-1. The surface was regenerated by washing with 0.5 m NaCl and 0.1 m HCl. Pull-down Experiments—In heparin pull-down experiments, 2 μg of glycosylated and deglycosylated FGFR1-ST in 0.5 ml of PBS was incubated overnight at 4 °C with 5 μl of heparin-agarose (Bio-Rad). Beads were collected by centrifugation at 13,000 × g for 3 min and washed twice with PBS by centrifugation. Protein in the supernatant was concentrated by freeze-drying. Supernatants and final pellets were adjusted in volume and proteins were analyzed by SDS-PAGE followed by silver nitrate staining. Analysis of the N-Glycans on FGFR1-ST—FGFR1-ST protein (50 μg) was reduced for 1 h at 37°C in 50 mm Tris-HCl buffer (pH 8.5) containing a 4-fold excess of dithiothreitol and carboxymethylated with a 2-fold molar excess of iodoacetic acid for 1 h at room temperature in the dark. Following dialysis at 4 °C for 72 h against 4 × 4.5 liters of cold 50 mm ammonium bicarbonate, pH 7.5, and lyophilization, FGFR1-ST was digested with trypsin (Thr1246, Sigma) at a 12:1 ratio (w/w) in 50 mm ammonium bicarbonate (pH 8.5) for 18 h at 37 °C. The reaction was stopped by adding a few drops of acetic acid to the solution. The sample was lyophilized prior to its dissolution in 150 μl (5% (v/v)) of acetic acid and purified using a SepPak cartridge C18 (Waters Corp), as described (35Sutton-Smith M. Dell A. Cell Biology: A Laboratory Handbook. Academic Press, San Diego2005Google Scholar). The purified glycopeptides were then digested with PNGase-F (Roche Applied Science, 1365177) in 50 mm ammonium bicarbonate (pH 8.5) containing 4.5 units of enzyme at 37 °C over 18 h. The sample was lyophilized, and the released N-glycans were purified using a SepPak cartridge C18 (Waters Corp) (35Sutton-Smith M. Dell A. Cell Biology: A Laboratory Handbook. Academic Press, San Diego2005Google Scholar). Permethylation and sample clean-up were performed using the sodium hydroxide protocol, as described previously (35Sutton-Smith M. Dell A. Cell Biology: A Laboratory Handbook. Academic Press, San Diego2005Google Scholar). MALDI-TOF data were acquired using a Perseptive Biosystems Voyager DE-STR™ mass spectrometer in the reflector mode with delayed extraction. MS/MS data were acquired using a 4800 MALDI TOF/TOF (Applied Biosystems) mass spectrometer. The collision energy was set to 1 kV, and air was used as collision gas. Samples were dissolved in 10 μl of methanol and mixed at a 1:1 ratio (v/v) with 2,5-dihydrobenzoic acid as matrix. In Silico Glycosylation of the FGF Receptor—In silico glycosylation of the FGF receptor using GlyProt (36Bohne-Lang A. von der Lieth C.W. Nucleic Acids Res. 2005; 33: 214-219Crossref PubMed Scopus (184) Google Scholar) employed two different models of the structures of immunoglobin loops D2 and D3 of FGFRs as input: the symmetric “two end” model of the FGFR1-IIIc·FGF-2·heparin hexasaccharide crystal structure (PDB ID: 1FQ9) and the asymmetric model of FGFR2-IIIc·FGF-1·heparin decasaccharide crystal structure (PDB ID: 1E0O). N-glycans were incorporated that correspond to structures identified by MALDI-TOF mass spectrometry. For a given model, the same glycan structure was used for all six potential N-glycosylation sites of the FGFR1-ST. PyMOL viewer software was used to visualized the model three-dimensional structures (DeLano, W. L. (2002) The PyMOL Molecular Graphics System). N-Glycosylation of Recombinant FGFR1 Proteins—Culture medium from CHO cells transfected with FGFR1-ST contained polypeptides immunoreactive to anti-His6 that migrated at an apparent molecular size of 50-70 kDa. When the same cells were cultured in the presence of tunicamycin (3 μg/ml), an inhibitor of N-glycosylation, a single immunoreactive band of 32 kDa was observed (Fig. 2A). Because the calculated molecular mass of FGFR1-ST is 31.5 kDa, this result suggested that the FGFR1-ST secreted by the transfected cells was heavily N-glycosylated. To determine if N-glycosylation was intrinsic to the protein or the consequence of it being secreted rather than membrane-bound, CHO cells were also transfected with FGFR1-GPI. In this case cell-associated immunoreactivity migrated as a series of bands between 45 and 75 kDa. When cultured in the presence of tunicamycin, cells expressed an immunoreactive product that migrated as a single band of 32 kDa, the expected size of FGFR1-GPI (Fig. 2A). FGFR1-ST was purified from CHO cell culture medium by nickel chelation and Strep-Tactin-Sepharose chromatography. After a subsequent separation by SDS-PAGE a series of bands between 50 and 75 kDa were revealed using silver staining. Preincubation of this material with N-glycanase, an enzyme which removes N-glycans, resulted in a single silver-stained band of 32 kDa (Fig. 2B). Thus, the multiple bands observed after separation of the FGFR1-ST reflect the existence of multiple glycoforms of the protein, which is pure as judged by silver staining. In cells treated with tunicamycin, an inhibitor of N-glycosylation, FGFR1-ST, and FGFR1-GPI still traversed the secretory pathway, because immunoreactive deglycosylated FGFR1-ST and FGFR1-GPI were detected in the culture medium and cells, respectively (Fig. 2A). The level of immunoreactive FGFR1-ST secreted into the culture medium is reduced in the presence of tunicamycin. This may reflect, at least in part, the inhibition of cell proliferation by tunicamycin and the fact that equivalent amounts of culture medium rather than cells are compared. 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